LE Waggoner et al. (1999) International C. elegans Meeting "A nicotinic acetylcholine receptor involved in regulation of egg-laying behavior."

DS Poole, WR Schafer, LE Waggoner

[International C. elegans Meeting, 1999]

We are interested in understanding the role nicotinic acetylcholine receptors play in egg-laying behavior. The nicotinic receptor agonist levamisole has been known to stimulate egg-laying in C. elegans . We have found that response is dependent on three genes, unc-29 , unc-38 and lev-1 , which encode nAChR subunits. In addition, mutations in these genes confer a slower rate of egg-laying under nominal conditions, suggesting that an UNC-29-containing nAChR promotes normal egg-laying. Expression studies using GFP-tagged UNC-29 show that this receptor is localized to both the vulval muscles and the VC neurons; however, it is not found in the HSNs. In addition, mosaic analysis, performed in our lab, indicates that while expression in both muscle and neurons is necessary for optimal response, expression in either location is sufficient for some stimulation of egg-laying by levamisole. Ablation of VC neuronal precursor cells prevents response to levamisole, supporting the hypothesis that the receptor's site of action is, in part, neuronal. Furthermore, cha-1 animals, which have low levels of endogenous ACh, are resistant to stimulation by levamisole, suggesting that the neuronal function of the receptor might be to facilitate of ACh, thus stimulating muscle contraction and egg-laying. Interestingly, we find that mutations in genes that confer weak resistance to levamisole in body wall muscle, including lev-8 and lev-9 , also show resistance to stimulation of egg-laying. We are interested in characterizing these genes, which are thought to encode positive regulators of nAChR activity. Progress in cloning one of these genes, lev-9 , will be presented.

Waggoner LE et al. (1996) West Coast Worm Meeting "PROBING THE MOLECULAR BASIS OF EGG-LAYING"

Waggoner LE, Schafer WR

[West Coast Worm Meeting, 1996]

Egg-laying in C. elegans is controlled by the vulval and uterine muscles. Regulation of these muscles and egg-laying behavior involves synaptic input from the HSN and VC motor neurons, as well as upstream input from the sensory amphids. Previous studies in our lab have shown that at least one mutant with mislocalization of axons from VC 4 and VC 5 exhibit early egg laying behavior (egl-c); several other egl-c mutants are defective in proteins that are expressed in the VC's. To further understand the function of these neurons in the regulation of egg-laying behavior, we have initiated experiments that involve ablation of VC 4 and VC 5 as well as the entire class of VC's. Regulation of egg-laying behavior also involves neurons that function upstream of the HSN and VC motor neurons. For example, environmental stimuli affecting egg-laying are detected by the sensory neurons of the amphids, and strains containing mutations in genes such as daf-7, which affect amphid function, exhibit an egl phenotype. However, little is known about how this sensory information is processed and used to regulate egg-laying behavior. To investigate these processes, we have designed a screen for revertants of the egl phenotype of daf-7 mutants. These revertants should include mutations in genes which function in neurons downstream of the amphid sensory cells.

Waggoner LE et al. (2000) West Coast Worm Meeting "Genetic Analysis of Nicotine Adaptation in C. elegans."

Poole DS, Dickinson KA, Schafer WR, Kim JS, Waggoner LE

[West Coast Worm Meeting, 2000]

In many organisms, prolonged exposure to nicotine causes long-lasting changes in the abundance and functional activity of nicotinic receptors, processes thought to underlie nicotine addiction in humans. At present, the molecular basis for nicotine adaptation is poorly understood. We have begun using a genetic approach to identify molecules required for nicotine adaptation in the egg-laying circuitry of C. elegans. Acute exposure to nicotine or the nicotinic agonist levamisole have dramatic effects on behavior, including stimulation of egg-laying. These acute effects of nicotine on egg-laying require the activity of a nicotinic receptor containing subunits unc-29, unc-38, and lev-1, as mutations in these genes confer resistance to nicotine and levamisole. This receptor appears to function in the vulval muscle, since expression of an unc-29 transgene under a vulval muscle-specific promoter in unc-29 mutant animals restored egg-laying sensitivity to levamisole. Upon long-term nicotine treatment, wildtype worms undergo adaptation, and acquire a long-lasting loss of egg-laying sensitivity. This effect is at least partially mediated at the level of receptor abundance, as the expression of an UNC-29:GFP chimera was drastically reduced upon overnight treatment with nicotine. In order to identify molecules required for the long-term effects of nicotine, we examined known mutants and screened for new mutants with defects in adaptation. From the known mutants, tpa-1, a PKC homologue, was shown to be necessary for the control of UNC-29 receptor abundance, since tpa-1 mutants remained sensitive to levamisole and retained high UNC-29 receptor levels in the vulval muscles even after long-term nicotine treatment. We also identified a new gene, nic-1, in a screen for adaptation defective mutants. These mutants displayed hypersensitivity to nicotine and a defect in adaptation, as well as an unusual locomotive phenotype and a deficiency in male mating ability, all of which are consistent with a defect in cholinergic function in the neuromusculature. Currently, we are in the process of mapping nic-1 in order to clone it, and hope to discover the identity of the molecule encoded by this gene.

Waggoner LE et al. (1997) International C. elegans Meeting "NEURONS AND GENES THAT CONTROL THE TIMING OF EGG-LAYING EVENTS"

Schafer WR, Waggoner LE

[International C. elegans Meeting, 1997]

We have begun to investigate the molecular and neural mechanisms that control timing of egg-laying events in C. elegans. Although egg-laying has been well studied at the genetic level, previous studies have revealed little about the temporal pattern of egg-laying in individual animals and how this pattern is regulated by sensory cues. By tracking wild-type animals for long time periods and analyzing the intervals between egg-laying events, we have found that animals fluctuate between two behavioral states: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs accumulate in the uterus. Switching between states, as well as egg-laying within the active state, all model as independent Poisson processes whose rate constants determine the rate of egg-laying. Sensory cues, such as salt concentration, modulate this pattern of behavior: high salt concentrations slow the rate of egg-laying primarily by lengthening the inactive phase. What is the neural basis for the generation and modulation of this pattern? Ablation of the serotonergic HSNs caused a lengthening of the inactive phase and prevented modulation by sensory information. Ablation of the VC4 and VC5 neurons, which may be serotonergic, also caused an increase in the duration of the inactive state on NGM (although they caused an Egl-c phenotype on M9). Elimination of both the HSNs and the VCs caused an extreme defect in egg-laying, yet the egg-laying events that did occur were still clustered. Thus, we propose that serotonin released from the HSNs and VCs facilitates entry into the active phase but is not required to maintain the active phase. What is the molecular nature of the two behavioral states? Two G-proteins, encoded by goa-1 and egl-30, are expressed in the vulval muscles and are known to affect egg-laying. The pattern of egg-laying in goa-1 mutants suggested that they lack the inactive phase; thus GOA-1 may be required for the vulval muscles to be in the inactive state.

Waggoner LE et al. (1998) Neuron "Control of alternative behavioral states by serotonin in Caenorhabditis elegans."

Waggoner LE, Schafer WR, Schafer RW, Zhou GT

[Neuron, 1998]

Serotonin has been implicated in the regulation of a wide range of brain functions involving alternative behavioral states, including the control of mood, aggression, sex, and sleep. Here, we report that in the nematode Caenorhabditis elegans, serotonin controls a switch between two distinct, on/off states of egg-laying behavior. Through quantitative analysis of the temporal pattern of egg-laying events, we determined that egg laying can be modeled as a novel random process, in which animals fluctuate between discrete behavioral states: an active state, during which eggs are laid in clusters, and an inactive state, during which eggs are retained. Single-cell ablation experiments indicate that two pairs of motor neurons, HSNL/HSNR and VC4/VC5, can induce the active phase by releasing serotonin. These neurons also release acetylcholine, which appears to trigger individual egg-laying events within the active phase. Genetic experiments suggest that determination of the behavioral states observed for C. elegans egg laying may be mediated through protein kinase C-dependent (PKC-dependent) modulation of voltage-gated calcium channels.

Waggoner LE et al. (1998) West Coast Worm Meeting "Characterization of the cellular and molecular basis for regulation of egg-laying"

Waggoner LE, Schafer WR

[West Coast Worm Meeting, 1998]

We have been investigating the molecular and neural mechanisms that control the temporal pattern of egg-laying in C. elegans. In order to observe egg-laying over long periods of time, we used an automated tracking system to record egg-laying events and then analyzed the data to measure intervals between events. These studies have allowed us to construct a model for egg-laying in which the animals fluctuate between two behavioral states: an active state, during which eggs are laid in clusters, and an inactive state , during which eggs accumulate in the uterus. Switching between states, as well as egg-laying within the active state, all model as independent Poisson processes whose rate constants determine the rate of egg-laying. By analyzing the egg-laying patterns of mutants and ablated animals, we have identified neurons and neurotransmitters involved in determining the rate constants. Several lines of evidence indicate that serotonin controls the switch from the inactive to the active state. For example, analyses of the egg-laying pattern of bas-1 and cat-4 mutants, both defective in serotonin synthesis, show an increase in the length of the inactive state, but do not affect egg-laying within clusters. Ablation studies have shown that serotonin may be released mainly from the HSNs but possibly also the VC4/5 motor neurons. Intriguingly, ablation of both HSNs and VCs results in a severe Egl phenotype that can not be rescued by serotonin. This indicates that another neurotransmitter, that can be provided by either the HSNs or VCs, is necessary for egg-laying. One possibility is acetylcholine; in fact, addition of both serotonin and the nicotinic agonist, levamisole, to double ablated animals rescues egg-laying, while neither serotonin nor levamisole alone is sufficient. Furthermore, mutants defective in choline acetyltransferase show a decrease in egg-laying within the active phase but have normal intervals between active phases. By studying the egg-laying patterns of Egl mutants, we have begun to characterize signaling pathways that control these two parameters. Some mutants show patterns similar to cat-4 and HSN-: longer inactive phases but normal egg-laying in clusters. One example is tpa-1, which is mutant for PKC. Since also serotonin resistant, it might be involved in signaling downstream of serotonin. Other mutants show a different pattern- an absence of clustered egg-laying events, which indicates an involvement in maintaining the active phase. One example is egl-19, which is mutant for a voltage gated Ca+2 channel. Furthermore, egl-19 mutants are resistant to serotonin, suggesting them as a target of serotonin in the vulval muscles. One model supporting this data suggests that serotonin is acting to stimulate egg-laying through a PKC dependent phosphorylation of Ca+2 channels, which could potentiate the induction of contraction by acetylcholine.

Waggoner LE et al. (2000) Genetics "Effect of a neuropeptide gene on behavioral states in Caenorhabditis elegans egg-laying."

Hardaker LA, Golik S, Schafer WR, Waggoner LE

[Genetics, 2000]

Egg-laying behavior in the nematode Caenorhabditis elegans involves fluctuation between alternative behavioral states: an inactive state, during which eggs are retained in the uterus, and an active state, during which eggs are laid in bursts. We have found that the flp-1 gene, which encodes a group of structurally related neuropeptides, functions specifically to promote the switch from the inactive to the active egg-laying state. Recessive mutations in flp-1 caused a significant increase in the duration of the inactive phase, yet egg-laying within the active phase was normal. This pattern resembled that previously observed in mutants defective in the biosynthesis of serotonin, a neuromodulator implicated in induction of the active phase. Although flp-1 mutants were sensitive to stimulation of egg-laying by serotonin, the magnitude of their serotonin response was abnormally low. Thus, the flp-1-encoded peptides and serotonin function most likely function in concert to facilitate the onset of the active egg-laying phase. Interestingly, we observed that flp-1 is necessary for animals to down-regulate their rate of egg-laying in the absence of food. Because flp-1 is known to be expressed in interneurons that are postsynaptic to a variety of chemosensory cells, the FLP-1 peptides may function to regulate the activity of the egg-laying circuitry in response to sensory cues.

Waggoner LE et al. (2000) J Neurosci "Long-term nicotine adaptation in Caenorhabditis elegans involves PKC-dependent changes in nicotinic receptor abundance."

Miwa J, Tabuse Y, Waggoner LE, Dickinson KA, Poole DS, Schafer WR

[J Neurosci, 2000]

Chronic exposure to nicotine leads to long-term changes in both the abundance and activity of nicotinic acetylcholine receptors, processes thought to contribute to nicotine addiction. We have found that in Caenorhabditis elegans, prolonged nicotine treatment results in a long-lasting decrease in the abundance of nicotinic receptors that control egg-laying. In naive animals, acute exposure to cholinergic agonists led to the efficient stimulation of egg-laying, a response mediated by a nicotinic receptor functionally expressed in the vulval muscle cells. Overnight exposure to nicotine led to a specific and long-lasting change in egg-laying behavior, which rendered the nicotine-adapted animals insensitive to simulation of egg-laying by the nicotinic agonist and was accompanied by a promoter-independent reduction in receptor protein levels. Mutants defective in the gene tpa-1, which encodes a homolog of protein kinase C (PKC), failed to undergo adaptation to nicotine; after chronic nicotine exposure they remained sensitive to cholinergic agonists and retained high levels of receptor protein in the vulval muscles. These results suggest that PKC-dependent signaling pathways may promote nicotine adaptation via regulation of nicotinic receptor synthesis or degradation.

Nathalie Pujol et al. (2001) Worm Breeder's Gazette "Ver midi IV"

Jonathan Ewbank, Nathalie Pujol

[Worm Breeder's Gazette, 2001]

La quatrieme reunion annuelle des equipes francaises ayant un interet pour C. elegans se tiendra le Vendredi 23 fevrier 2001 a Luminy, Marseille. Contact: pujol@ibdm.univ-mrs.fr ewbank@ciml.univ-mrs.fr

Stanley I Shyn et al. (2001) International C. elegans Meeting "Calcium imaging of vulval muscle activity in egg-laying behavior"

William R Schafer, Stanley I Shyn

[International C. elegans Meeting, 2001]

Serotonin (5HT) is a neurotransmitter which often functions as a modulator of tissue excitability and behavioral states. In C. elegans , one behavioral effect it produces is the stimulation of egg-laying. 1 One mechanism by which it appears to do this is by shifting the worm's vulval muscles from a quiescent or inactive mode to a more active one during which eggs are laid in a cluster. Through a combination of genetic and pharmacologic approaches, we have identified mutants in several genes to be 5HT-resistant for this behavior: egl-19 (L-type voltage gated Ca 2+ channel alpha-1 subunit), tpa-1 (PKC homolog), acy-1 (adenylate cyclase), & gpa-14 (novel G-protein). 2,3 While implicated in 5HT-signalling, these molecules may also constitute effectors from other neurotransmitter systems known to interact with 5HT in egg-laying, including neuropeptide signalling: Neuropeptides derived from the flp-1 gene, for example, are known to potentiate 5HT-response. 4,5 To gain more insight into these issues, we have been utilizing Cameleon, a genetically encodable and ratiometric Ca 2+ -sensor 6,7 in both intact & cut-worm preparations to study how vulval muscle physiology changes in the presence or absence of 5HT & and how it responds to agents like forskolin or FLP peptides in N2s and in various mutant backgrounds. Preliminary results indicate that, in the absence of 5HT, vulval muscles in N2 worms typically show sporadic but often clustered Ca 2+ transients. Upon application of exogenous 5HT (5 mg/ml), this pattern gives way to a more rhythmic train of small transient events (~0.5 Hz). This data, along with initial mutant characterization, will be presented at the June meeting. 1 Horvitz HR et al , Science 216 :1012-4 2 Waggoner LE et al , Neuron 21 :203-214 3 Shyn S & Schafer W, 2000 WCWM abstract 228 4 Schinkmann K & Li C, J Comp Neurol 316 :251-260 5 Waggoner LE et al , Genetics 154 :1181-1192 6 Miyawaki A et al , Nature 388 :882-887 7 Kerr R et al , Neuron 26 :583-594